This invention relates to the use of milk which is free of the xcex2-casein A1 protein in the prevention or treatment of coronary heart disease. The invention also relates to the testing of DNA from cells obtained from lactating bovines for the presence of DNA encoding certain xcex2-casein proteins, selecting the bovines on the basis of the testing, and then milking those bovines to produce milk free of xcex2-casein A1 for use in the prevention or treatment of coronary heart disease.
Coronary heart disease is a major cause of death, particularly in countries where the populations are well-nourished, such as in the western world. Many factors are implicated as risk factors for this disease including obesity, smoking, genetic predisposition, diet, hypertension, and cholesterol.
Dairy products, especially milk, are a major contributor to the dietary intake of humans, again particularly in western world populations. Milk contains numerous components of nutritional and health benefit. Calcium is one example. However, milk is also a significant source of dietary fat. It is widely accepted that saturated fats found in milk are a risk factor for coronary heart disease. However, the inventor has discovered an additional risk factor present in some bovine milk unrelated to the fat content. What is entirely surprising is the source of the risk. The source is not dependent on the fat content of milk. Instead, it is a milk protein, xcex2-casein, which is linked to coronary heart disease.
A number of variants of milk proteins have been identified. Initially, three variants of xcex2-casein were discovered (Aschaffenburg, 1961) and were denoted A, B and C. It was later found that the A variant could be resolved into A1, A2 and A3 by gel electrophoresis (Peterson et. al. 1966). The xcex2-casein variants now known are A1, A2, A3, B, C, D, E and F, with A1 and A2 being present in milk in the highest proportions. It is anticipated that other variants may be identified in the future.
The inventor has determined that it is the milk protein xcex2-casein A1 which represents the risk factor in bovine milk that is linked to coronary heart disease, or at least is the principal risk factor. This determination on the part of the inventor forms the basis of the present invention.
There is no relationship between the fat content of milk and xcex2-casein genotype in cows. Therefore, selecting cattle on the basis of milk fat content will not identify which bovines produce the novel risk factor, namely the specific xcex2-casein variant, in their milk.
There is no significant difference in the fat content of milk produced by cows which are homozygous for the xcex2-casein A1 allele (i.e. A1A1) and cows which are homozygous for the xcex2-casein A2 allele (i.e. A2A2). This is apparent from studies reported in the literature.
Ng-Kwai-Hang has carried out several studies. One study (Ng-Kwai-Hang et. al., 1990) suggested that milk containing xcex2-casein A1 rather than xcex2-casein A2 may have a slightly higher fat content. However, these differences were very small. The differences between milk from A1 homozygous cows and milk from A2 homozygous cows were 0.05% (for the first lactation period), 0.07% (for the second lactation period), and 0.04% (for the third lactation period).
In another study, Ng-Kwai-Hang (in an abstract cited by Jakob et. al;, 1990) found the opposite effect (i.e. the A1A1 product had a lower fat content than the A2A2 product). Thus, the 1995 Ng-Kwai-Hang abstract directly contradicts the Ng-Kwai-Hang, et. al., 1990 study.
McLean et. al., 1984 (McLean) also reported that there was no significant difference in the fat content of milk from cows of A1A1 and A2A2 genotypes (meanxc2x1standard error: 45.8xc2x12.6 g/l for milk of A1A1 cows and 48.6xc2x11.9 g/l for milk of A2A2 cows).
Aleandri et. al., 1990 (Aleandri), shows in Table 5 that the least squares estimates of the effects of different genotypes and their standard errors on fat percentage in milk are 0.12xc2x10.09 for A1A1 cows and 0.07xc2x10.09 for A2A2 cows. Taking into account the standard error for the test, Aleandri indicates that the effects of A1A1 and A2A2 genotypes on milk fat content are equivalent.
Bovenhuis et. al., 1992 (Bovenhuis), highlights that there are statistical problems associated with the way in which the genotype effects on fat percentages in milk are studied and documented. It is stated that the ordinary least squares estimates may be biased. Bovenhuis points out that the analysis of the effect of a particular genotype on various characteristics of milk is complex in nature and may, among other things, be affected by other genes which may be linked to the gene under study. Bovenhuis attempts to take into account the above variables and to overcome statistical problems by using an animal model method.
Table 3 of Bovenhuis indicates that, for a statistical model in which each milk protein gene is analysed separately and the A1A1 cows designated as being the standard (i.e. given a value of 0% fat attributable to the genotype), the A2A2 genotype was estimated not to contribute (i.e. 0%) to the fat content of the milk of the animals harbouring that genotype when compared to the A1A1 genotype. The standard error of the test is recorded as 0.02%. Where a statistical model was used in which all milk protein genes were analysed simultaneously (Table 4 of Bovenhuis) and the A1A1 genotype was again designated as being the standard (at 0% fat content attributable to the A1A1 genotype), the A2A2 genotype was estimated to contribute to the fat content of the milk at xe2x88x920.01% when compared with the A1A1 genotype. In this study a standard error of 0.02 was designated. Taking into account the standard error of the tests these results indicate that the A2A2 genotype contributes to the fat content of milk in an equivalent manner to the genotype A1A1.
Gonyon et. al., 1987 reached the same conclusion as Bovenhuis.
The level of individual components in milk is influenced by both the genotype and the environment. That is, the variation between animals in milk output or milk composition is due to both genotypic and phenotypic factors. For example, Bassette et. al., 1988 (Bassette) indicates that the composition of bovine milk may be influenced by a number of environmental factors and conditions other than genetic factors. Environmental factors may impact on milk production and the constituents contained within the milk (including fat content). For example, changes in milk composition occur due to:
the stage of lactation (e.g., the fat content of colostrum is often higher; the concentration of fat changes over a period of many weeks as the cow goes through lactation);
the age of the cow and the number of previous lactations;
the nutrition of the cow including the type and composition of feed consumed by the cow;
seasonal variations;
the environmental temperature at which the cows are held;
variations due to the milking procedure (e.g., the fat content of milk tends to increase during the milking process which means that for an incomplete milking the fat content would generally be lower than normal and for a complete milking the fat content will be higher than normal); and
milking at different times of the day.
It is therefore apparent from the studies in this field that a person skilled in the relevant area of technology would not find a link between the fat content of milk and the xcex2-casein genotype of the milk-producing bovines from which that milk is produced.
Thus, the inventor has for the first time identified the milk protein xcex2-casein A1 as a risk factor linked to coronary heart disease in its own right. It is therefore an object of this invention to provide a method of using milk substantially free of xcex2-casein A1 to prevent or treat coronary heart disease or to minimise the risk of developing coronary heart disease, or to at least provide a useful alternative. It is also an object of the invention to provide a method of producing milk substantially free of xcex2-casein A1 suitable for use in the prevention or treatment of coronary heart disease or the minimisation of the risk of developing coronary heart disease, or to at least provide a useful alternative.
In one aspect of the invention there is provided a method of preventing or treating coronary heart disease in a human which includes the step of at least reducing the intake in that human of xcex2-casein A1.
Preferably the reduction is effected by the human ingesting milk obtained from one or more lactating bovines, or a product processed from that milk, where the milk or product ingested is substantially free of xcex2-casein A1.
Preferably the milk is substantially free of xcex2casein A1 but contains any one or more of xcex2-caseins A2, A3, B. C, D and E.
More preferably the milk is substantially free of xcex2-caseins A1, B and C but contains any one or more of xcex2-caseins A2, A3, D and E. Most preferably the milk is substantially free of xcex2-caseins A1, B and C and contains only xcex2-casein A2.
In a second aspect of the invention there is provided a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more lactating bovines which milk is substantially free of xcex2-casein A1 but which contains any one or more of xcex2-caseins A2, A3, B, C, D and E, the method including the steps of:
(i) testing DNA or RNA from cells containing DNA or RNA obtained from the one or more lactating bovines for the presence of DNA or RNA encoding xcex2-casein A1;
(ii) selecting bovines which do not have any DNA or RNA encoding xcex2-casein A1; and
(iii) milking the selected bovines.
In another aspect of the invention there is provided a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more lactating bovines which milk is substantially free of xcex2-casein A1, B and C but which contains any one or more of xcex2-caseins A2, A3, D and E, the method including the steps of:
(i) testing DNA or RNA from cells containing DNA or RNA obtained from the one or more lactating bovines for the presence of DNA or RNA encoding any one or more of xcex2-caseins A1, B and C;
(ii) selecting bovines which do not have any DNA or RNA encoding any one or more of xcex2-caseins A1, B and C; and
(iii) milking the selected bovines
In another aspect of the invention there is provided a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more lactating bovines which milk is substantially free of xcex2-casein A1 but which contains xcex2-casein A2, the method including the steps of:
(i) testing DNA or RNA from cells containing DNA or RNA obtained from the one or more lactating bovines for the presence of DNA or RNA encoding xcex2-casein A2;
(ii) selecting bovines which are homozygous for DNA or RNA encoding xcex2-casein A2; and
(iii) milking the selected bovines.
In another aspect of the invention there is provided a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more lactating bovines which milk is substantially free of xcex2-casein A1 but which contains any one or more of xcex2-caseins A2, A3, D and E, the method including the steps of:
(i) testing DNA or RNA from cells containing DNA or RNA obtained from the one or more lactating bovines for the presence of DNA or RNA encoding any one or more of xcex2-caseins A2, A3, D and E;
(ii) selecting bovines which have DNA or RNA encoding only for any one or more of xcex2caseins A2, A3, D and E; and
(iii) milking the selected bovines.
In another aspect of the invention there is a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more lactating bovines which milk is substantially free of xcex2-casein A1 but which contains xcex2-casein A2, the method including the steps of:
(i) testing DNA or RNA from cells containing DNA or RNA obtained from the one or more lactating bovines for the presence of DNA or RNA encoding xcex2-casein A1 and DNA or RNA encoding xcex2-casein A2;
(ii) separating bovines which are homozygous for DNA or RNA encoding xcex2-casein A2 from bovines which either have DNA or RNA encoding xcex2-casein A1 or which have DNA or RNA encoding both 3-casein A1 and xcex2-casein A2; and
(iii) milking the bovines which are homozygous for DNA or RNA encoding xcex2-casein A2.
In another aspect of the invention there is provided a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more lactating bovines which milk is substantially free of xcex2-caseins A1, B and C but which contains any one or more of xcex2-caseins A2, A3, D and E, the method including the steps of:
(i) testing DNA or RNA from cells containing DNA or RNA obtained from the one or more lactating bovines for the presence of DNA or RNA encoding any one or more of xcex2-caseins A1, B and C and DNA or RNA encoding any one or more of xcex2-caseins A2, A3, D and E.
(ii) separating bovines which have any DNA or RNA encoding any one or more of xcex2-caseins A1, B and C from bovines which have DNA or RNA encoding only for any one or more of xcex2-caseins A2, A3, D and E; and
(iii) milking the bovines which have DNA or RNA encoding only for any one or more of xcex2-caseins A2, A3, D and E.
Preferably the one or more lactating bovines of any aspect of this invention are Bos taurus bovines
More preferably the milk produced according to any aspect of this invention is substantially free of xcex2-casein A1 and the xcex2-casein contained in the milk comprises greater than 95% by weight xcex2-casein A2.
More preferably the milk produced according to any aspect of this invention is substantially free of xcex2-casein A1 and the xcex2-casein contained in the milk comprises approximately 100% by weight xcex2-casein A2.